As hypusine biosynthetic steps present unique target for intervention in aberrant cell proliferation, we need to develop new inhibitors of deoxyhypusine synthase (DHS) through high throughput screening (HTS) of small molecule libraries. However, the conventional DHS assay measuring incorporation of radioactivity from spermidine into eIF5A protein is not suitable for HTS. In this period, we have concentrated our efforts on the development of a new DHS assay that is compatible with HTS. This was accomplished by incubation of DHS with spermidine and NAD in the absence of eIF5A precursor protein to generate NADH and by detection of NADH using NADH-Glo assay. Robust luminescence signals were generated in the DHS/NADH-Glo coupled reaction. The reaction is sensitive and reproducible. Using this assay, we have initiated HTS and will continue screening of several chemical libraries. Since HTS requires a large amount of DHS, we also devised a new method to purify recombinant DHS protein from E. coli BL21(DE3) cells in high yield, by codon optimization. Although majority of the recombinant enzymes resulted in inclusion bodies, the enzyme was solubilized from inclusion bodies using high pH Tris buffer containing 2M urea.